A Novel Agonist of the TRIF Pathway Induces a Cellular State Refractory to Replication of Zika, Chikungunya, and Dengue Viruses

Abstract

The ongoing concurrent outbreaks of Zika, Chikungunya, and dengue viruses in Latin America and the Caribbean highlight the need for development of broad-spectrum antiviral treatments. The type I interferon (IFN) system has evolved in vertebrates to generate tissue responses that actively block replication of multiple known and potentially zoonotic viruses. As such, its control and activation through pharmacological agents may represent a novel therapeutic strategy for simultaneously impairing growth of multiple virus types and rendering host populations resistant to virus spread. In light of this strategy's potential, we undertook a screen to identify novel interferon-activating small molecules. Here, we describe 1-(2-fluorophenyl)-2-(5-isopropyl-1,3,4-thiadiazol-2-yl)-1,2-dihydrochromeno[2,3-c]pyrrole-3,9-dione, which we termed AV-C. Treatment of human cells with AV-C activates innate and interferon-associated responses that strongly inhibit replication of Zika, Chikungunya, and dengue viruses. By utilizing genome editing, we investigated the host proteins essential to AV-C-induced cellular states. This showed that the compound requires a TRIF-dependent signaling cascade that culminates in IFN regulatory factor 3 (IRF3)-dependent expression and secretion of type I interferon to elicit antiviral responses. The other canonical IRF3-terminal adaptor proteins STING and IPS-1/MAVS were dispensable for AV-C-induced phenotypes. However, our work revealed an important inhibitory role for IPS-1/MAVS, but not TRIF, in flavivirus replication, implying that TRIF-directed viral evasion may not occur. Additionally, we show that in response to AV-C, primary human peripheral blood mononuclear cells secrete proinflammatory cytokines that are linked with establishment of adaptive immunity to viral pathogens. Ultimately, synthetic innate immune activators such as AV-C may serve multiple therapeutic purposes, including direct antimicrobial responses and facilitation of pathogen-directed adaptive immunity.IMPORTANCE The type I interferon system is part of the innate immune response that has evolved in vertebrates as a first line of broad-spectrum immunological defense against an unknowable diversity of microbial, especially viral, pathogens. Here, we characterize a novel small molecule that artificially activates this response and in so doing generates a cellular state antagonistic to growth of currently emerging viruses: Zika virus, Chikungunya virus, and dengue virus. We also show that this molecule is capable of eliciting cellular responses that are predictive of establishment of adaptive immunity. As such, this agent may represent a powerful and multipronged therapeutic tool to combat emerging and other viral diseases.

Keywords: antiviral agents; emerging virus; innate immunity; interferons.

FIG 1

Activation of type I interferon-associated transcription after exposure to AV-C. (A) Chemical structure of 1-(2-fluorophenyl)-2-(5-isopropyl-1,3,4-thiadiazol-2-yl)-1,2-dihydrochromeno[2,3-c]pyrrole-3,9-dione (AV-C). (B) Reporter assay results, showing induction of ISRE-dependent LUC expression in THF-ISRE cells at 8 h posttreatment at the indicated concentrations. Values displayed are average fold changes ± standard deviations, based on three replicates in comparison to results in DMSO-treated cells. Unpaired-sample Student’s t test comparisons were made between AV-C- and DMSO-treated cells. **, P ≤ 0.01; ***, P ≤ 0.001.

FIG 2

AV-C-mediated induction of IFN-stimulated antiviral mRNA and proteins. (A) Heat map illustrating signals from individual hybridization array probes relative to the mean composite signal for all treatments of THF cells (untreated mock, 1,000 U/ml IFN-β, or 25 μM AV-C). Data presented are expressed as the log₂ fold difference, and only probes found to be significantly increased or decreased following any treatment are presented. (B) Venn diagram illustrating numbers of annotated RNAs significantly up- or downregulated relative to results with mock-treated cells for AV-C- or IFN-β-treated cells. (C) Immunoblot results for IFN-stimulated antiviral proteins IFIT1 and Mx1 in THFs left untreated or treated with AV-C (25 μM) or IFN-β (1,000 U/ml) for 8 h.

FIG 3

AV-C elicits a cellular state refractory to virus replication. Average results (PFU per milliliter, ± the standard deviation) of ZIKV, CHIKV, and DENV grown on THF cells in triplicate in the presence of the indicated AV-C concentrations (the DMSO concentration was normalized to 1%) added 6 h preinfection. Unpaired-sample Student’s t test comparisons were made between AV-C- and DMSO-treated cells. **, P ≤ 0.01; ***, P ≤ 0.001.

FIG 4

AV-C induces canonical IRF3 activation and IRF3- and IFN-dependent transcription. (A) Immunoblot results, showing S386 phosphorylation status of IRF3, total IRF3, and GAPDH from THF lysates following 4-h treatment with 1% DMSO (untreated), 100 μM G10, 1 ng/ml LPS, 160 HAU/ml SeV, or 25 μM AV-C. (B) Reporter assay results, showing induction of ISRE-dependent LUC expression in THF-ISRE cells lacking IRF3 following 8-h treatment with UV-inactivated HCMV (MOI, 3), 1,000 U/ml IFN-β, or AV-C at the indicated concentrations. Values displayed are average fold changes versus results in DMSO-treated cells of three replicates, ± the standard deviation (SD). (C) Transcription of ISG15, IFIT1, and Mx2 in cells treated for 8 h with 25 μM AV-C, SeV (160 HAU/ml), or 1,000 U/ml IFN-β. Values displayed are average fold changes versus results in DMSO-treated cells of two biological replicates, ± SD. (D) Reporter assay showing induction of LUC expression in THF-ISRE cells left untreated or exposed to UV-HCMV (MOI, 3) or 25 μM AV-C in the presence or absence of the TBK1/IKKi inhibitor BX795 (10 nM). Values displayed are average fold changes versus results with DMSO-treated cells of three replicates, ± SD. (E) Immunoblot results, showing the S386 phosphorylation status of IRF3 as well as total IRF3 in THFs left untreated following 4-h exposure to UV-HCMV (MOI, 3) or 25 μM AV-C in the presence or absence of 10 nM BX795.

FIG 5

AV-C induces IRF3-mediated transcriptional activity in a manner independent of STING and IPS-1/MAVS but dependent on TRIF. (A) Reporter assay results, showing induction of ISRE-dependent LUC expression in THF-ISRE cells lacking IPS1/MAVS (THF-ISRE-ΔIPS1) following 8-h treatment with SeV (160 HAU/ml) or AV-C at the indicated concentrations. Values displayed are average fold changes compared to results with DMSO-treated cells of three replicates (± standard deviations [SD]). (B) Transcription of ISG15, IFIT1, viperin, and Mx2 in THF-ISRE-ΔIPS1 cells treated for 8 h with SeV (160 HAU/ml), or 50 μM AV-C. Values displayed are average fold changes versus results in DMSO-treated cells of two biological replicates, ± SD. (C) Immunoblot showing S386 phosphorylation status of IRF3 as well as total IRF3, TRIF, STING, IPS1/MAVS, and GAPDH in THF-ISRE-ΔIPS1 cells left untreated and following 4-h exposure to 100 μM G10, SeV (160 HAU/ml), or 25 μM AV-C. (D) Reporter assay results, showing induction of ISRE-dependent LUC expression in THF-ISRE cells lacking STING (THF-ISRE-ΔSTING) following 8-h treatment with 100 μM G10 or AV-C at the indicated concentrations. Values displayed are average fold changes compared to results in DMSO-treated cells of three replicates, ± SD. (E) Transcription of ISG15, IFIT1, viperin, and Mx2 in THF-ISRE-ΔSTING cells treated for 8 h with 100 μM G10 or 25 μM AV-C. Values displayed are average fold changes compared to results in DMSO-treated cells of two biological replicates, ± SD. (F) Immunoblot results showing the S386 phosphorylation status of IRF3 as well as total IRF3, TRIF, STING, IPS1/MAVS, and GAPDH in THF-ISRE-ΔSTING cells left untreated and following 4-h exposure to 100 μM G10 or 25 μM AV-C. (G) Reporter assay results, showing induction of ISRE-dependent LUC expression in THF-ISRE cells lacking TRIF (THF-ISRE-ΔTRIF) following 8-h treatment with 100 μM G10 or AV-C at the indicated concentrations. Values displayed are average fold changes compared to results in DMSO-treated cells of three replicates, ± SD. (H) Transcription of ISG15, IFIT1, viperin, and Mx2 in THF-ISRE-ΔTRIF cells treated for 8 h with 100 μM G10 or 25 μM AV-C. Values displayed are average fold changes versus results in DMSO-treated cells of two biological replicates, ± SD. (I) Immunoblot results, showing S386 phosphorylation status of IRF3 as well as total IRF3, TRIF, STING, IPS1/MAVS, and GAPDH in THF-ISRE-ΔTRIF cells left untreated and following 4-h exposure to 100 μM G10 or 25 μM AV-C.

FIG 6

AV-C elicits an antiviral state in cells lacking the IPS-1/MAVS or STING pathways but not the TRIF or IFNAR pathways. (A) Average PFU per milliliter (± the standard deviation) for CHIKV, DENV, or ZIKV grown on wild-type (WT) THF-ISRE cells (blue) and THF-ISRE lacking IPS-1/MAVS (red), STING (gray), TRIF (black), or IFNAR (yellow) following 6 h pretreatment with 1% DMSO, 12.5 μM AV-C, or 1,000 U/ml IFN-β (the DMSO concentration was normalized to 1%). Infections were performed in triplicate, and virus titers were determined by serial dilution plaque assay at 48 h postinfection (CHIKV) or by FFU assay at 72 h postinfection (ZIKV and DENV) as described in the text. (B) Average units per milliliter of type I IFN, as determined in a luciferase bioassay of levels secreted from the indicated cells following 8-h treatment with 12.5 μM AV-C. (C) Average (in picograms per milliliter) ± the standard deviation of secreted IFN-β, determined by ELISA from human PBMCs following 8-h treatment in triplicate with the indicated concentrations of AV-C. An unpaired Student’s t test was used to determine statistical significance. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 7

AV-C-mediated innate activation of primary human peripheral blood mononuclear cells. (A) Immunoblot showing the S386 phosphorylation status of IRF3, total IRF3, and GAPDH from human PBMC lysates following 4-h treatment with 1% DMSO or 12.5 μM AV-C. (B) Transcription of IFIT1, viperin, Mx2, IL-6, and IL-1β in PBMCs treated for 8 h with 1 μg/ml LPS, 12.5 μM AV-C, or 25 μM AV-C. Values displayed are average fold changes versus results in DMSO-treated cells of two biological replicates, ± standard deviations. (C) Secretion of TNF-α, IL-6, and IL-1β from PBMCs either left untreated (NT) or exposed for 24 h to 100 ng/ml LPS or 25 μM AV-C. Values presented are averages (in picograms per milliliter) ± the standard deviation from cells from five individual donors (donor-specific values are color coded). Paired-sample Student’s t test comparisons were made between treated and NT cells. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 8

NF-κB activity is not induced by exposure to AV-C. (A) Reporter assay results, showing induction of NF-κB-dependent LUC expression in THF–NF-κB cells at 8 h posttreatment with 160 HAU/ml SeV, 10 ng/ml TNF-α, or the indicated concentration of AV-C. Values displayed are average fold changes (± standard deviations) based on three replicates compared to DMSO-treated cells. (B) Subcellular localization of NF-κB subunit P65 in THF cells following 4-h exposure to 1% DMSO, 160 HAU/ml SeV, or 25 μM AV-C. (C) Degradation of IκBα in PBMCs from two donors following exposure to 1% DMSO, 12.5 μM AV-C, or 100 ng/ml LPS for the indicated time (β-actin served as the loading control).